Project Summary Vibrio cholerae causes the severe diarrheal disease cholera that is endemic in much of Asia, Africa, and South America, and has recently been reintroduced into Haiti. The species is highly diverse, although only O1 or O139 serogroup strains cause epidemic disease. However, increasing sporadic disease has been reported in endemic areas, and is caused by strains belonging to non-O1/non-O139 serogroups that present a public health threat both in developed and industrialized nations, including the United States. Unlike pathogenic O1 and O139 strains, the vast majority of pathogenic non-O1/non-O139 strains do not carry the well characterized virulence factors for colonization (TCP) and toxin production (CT), and the virulence mechanisms used by these strains are not well understood. Our study of pathogenic non-O1/non-O139 serogroup strains began with genomic sequencing of the clinically isolated O39 serogroup strain, AM-19226, which revealed a Type Three Secretion System (T3SS) that is conserved among other V. cholerae isolates. Our subsequent experiments identified 13 effector proteins that are translocated into the eukaryotic cell by the T3SS apparatus, and two transcriptional regulators encoded within the T3SS genomic island. We hypothesize that a subset of effectors and a T3SS encoded non-effector protein play important roles in colonization, and that other, unique V. cholerae effectors cooperate to disrupt host cell signaling to result in the diarrheal response that arises from cell-cell junction disruption, ionic transport imbalance and/or cellular stress responses. We propose to use complimentary in vitro and in vivo approaches to identify and characterize the mechanism of colonization and the host cell proteins and pathways targeted by V. cholerae effector proteins. Initial studies will define the minimum set of T3SS encoded genes necessary for colonization and define their roles in adherence to host cells. Effector protein analysis will focus on a total of five proteins and their roles in different stages of infection/disease: VopZZ, VopX, VopM, VopF, and VopK. VopZZ and VopM are critical for colonization, and Vops F and M have functions associated with cytoskeletal remodeling, which will be investigated in our studies. VopsX and K are unique to the V. cholerae T3SS, VopsX and K. We will use our experience the S. cerevisiae model system to discover host cell proteins that are the targets of effector activity and direct our studies in mammalian cells. We will also examine the host cell response to AM-19226 infection in vitro using mammalian co-culture and expression models. The co-culture assay will be used as a tool to dissect how effectors interact with mammalian signal transduction pathways during infection to disrupt homeostasis, whereas a viral-based expression system will be used to biochemically identify targets of effector protein activity and the response of mammalian cells to effector expression. We expect our results to reveal the molecular mechanisms of TCP/CT independent pathogenesis in the subset of non-O1/non-O139 strains that encode a T3SS.